Biofertilizers can enhance nitrogen use efficiency of sugarcane.

Fertilizers are costly inputs into crop systems. To compensate for inefficiencies and losses from soil, farmers apply on average double the amount of nitrogen (N) fertilizer acquired by crops. We explored if N efficiency improves with biofertilizers formulated with organic waste, mineral N or plant growth-promoting rhizobacteria (PGPR). We compared treatments receiving mineral N fertilizer or biofertilizers at industry-recommended (100%) or lower (60%) N rates at two commercial sugarcane farms. Biofertilizer at the 60% N-rate generated promising results at one farm with significantly higher biomass and sugar yield than the no-N control, which matched the 100% mineral N treatment. This yield difference was accompanied by a shift in microbial diversity and composition. Correlation analysis confirmed that shifts in microbial communities were strongly linked to soil mineral N levels, as well as crop productivity and yield. Microbial co-occurrence networks further revealed that biofertilizer, including treatments with an added PGPR, can enhance bacterial associations, especially in the context of complex fungal networks. Collectively, the results confirm that biofertilizers have quantifiable effects on soil microbial communities in a crop system setting, which underscores the opportunities for biofertilizers to promote N use efficiency and the circular N economy.

Plant growth promoting rhizobacteria increase the efficiency of fertilisers while reducing nitrogen loss.

More than half of the applied conventional fertiliser nitrogen (N) in cropping systems can be lost to the environment, resulting in water and air pollution. Farming systems that ensure efficient fertiliser use are crucial to sustain crop productivity without harming the environment. One avenue to achieve this is the use of bio-fertilisers with recognised benefits for plant nutrition and soil heath. Within this area, plant growth promoting rhizobacteria (PGPR) are increasingly applied to enhance plant nutrient acquisition and assimilation. Here, we investigated if PGPR can improve fertiliser performance. We show that the addition of PGPR to soils amended with 50% organic and 50% conventional N fertilisers increased the growth of kikuyu grass (Pennisetum clandestinum), producing yields similar to those obtained using 100% conventional N fertiliser. Encouragingly, this combination also reduced mineral N leaching by 95% relative to the all conventional fertiliser treatment. These findings suggest that using organic and synthetic fertilisers together in the presence of PGPR is a promising approach for sustaining plant growth while reducing potential pollution from inefficient use of conventional N fertilisers.

The core root microbiome of sugarcanes cultivated under varying nitrogen fertilizer application.

Diazotrophic bacteria potentially supply substantial amounts of biologically fixed nitrogen to crops, but their occurrence may be suppressed by high nitrogen fertilizer application. Here, we explored the impact of high nitrogen fertilizer rates on the presence of diazotrophs in field-grown sugarcane with industry-standard or reduced nitrogen fertilizer application. Despite large differences in soil microbial communities between test sites, a core sugarcane root microbiome was identified. The sugarcane root-enriched core taxa overlap with those of Arabidopsis thaliana raising the possibility that certain bacterial families have had long association with plants. Reduced nitrogen fertilizer application had remarkably little effect on the core root microbiome and did not increase the relative abundance of root-associated diazotrophs or nif gene counts. Correspondingly, low nitrogen fertilizer crops had lower biomass and nitrogen content, reflecting a lack of major input of biologically fixed nitrogen, indicating that manipulating nitrogen fertilizer rates does not improve sugarcane yields by enriching diazotrophic populations under the test conditions. Standard nitrogen fertilizer crops had improved biomass and nitrogen content, and corresponding soils had higher abundances of nitrification and denitrification genes. These findings highlight that achieving a balance in maximizing crop yields and minimizing nutrient pollution associated with nitrogen fertilizer application requires understanding of how microbial communities respond to fertilizer use.

DNA is taken up by root hairs and pollen, and stimulates root and pollen tube growth.

Phosphorus (P) enters roots as inorganic phosphate (P(i)) derived from organic and inorganic P compounds in the soil. Nucleic acids can support plant growth as the sole source of P in axenic culture but are thought to be converted into P(i) by plant-derived nucleases and phosphatases prior to uptake. Here, we show that a nuclease-resistant analog of DNA is taken up by plant cells. Fluorescently labeled S-DNA of 25 bp, which is protected against enzymatic breakdown by its phosphorothioate backbone, was taken up and detected in root cells including root hairs and pollen tubes. These results indicate that current views of plant P acquisition may have to be revised to include uptake of DNA into cells. We further show that addition of DNA to P(i)-containing growth medium enhanced the growth of lateral roots and root hairs even though plants were P replete and had similar biomass as plants supplied with P(i) only. Exogenously supplied DNA increased length growth of pollen tubes, which were studied because they have similar elongated and polarized growth as root hairs. Our results indicate that DNA is not only taken up and used as a P source by plants, but ironically and independent of P(i) supply, DNA also induces morphological changes in roots similar to those observed with P limitation. This study provides, to our knowledge, first evidence that exogenous DNA could act nonspecifically as signaling molecules for root development.

Plants can use protein as a nitrogen source without assistance from other organisms.

Nitrogen is quantitatively the most important nutrient that plants acquire from the soil. It is well established that plant roots take up nitrogen compounds of low molecular mass, including ammonium, nitrate, and amino acids. However, in the soil of natural ecosystems, nitrogen occurs predominantly as proteins. This complex organic form of nitrogen is considered to be not directly available to plants. We examined the long-held view that plants depend on specialized symbioses with fungi (mycorrhizas) to access soil protein and studied the woody heathland plant Hakea actites and the herbaceous model plant Arabidopsis thaliana, which do not form mycorrhizas. We show that both species can use protein as a nitrogen source for growth without assistance from other organisms. We identified two mechanisms by which roots access protein. Roots exude proteolytic enzymes that digest protein at the root surface and possibly in the apoplast of the root cortex. Intact protein also was taken up into root cells most likely via endocytosis. These findings change our view of the spectrum of nitrogen sources that plants can access and challenge the current paradigm that plants rely on microbes and soil fauna for the breakdown of organic matter.

Nitrogen affects cluster root formation and expression of putative peptide transporters.

Non-mycorrhizal Hakea actites (Proteaceae) grows in heathland where organic nitrogen (ON) dominates the soil nitrogen (N) pool. Hakea actites uses ON for growth, but the role of cluster roots in ON acquisition is unknown. The aim of the present study was to ascertain how N form and concentration affect cluster root formation and expression of peptide transporters. Hydroponically grown plants produced most biomass with low molecular weight ON>inorganic N>high molecular weight ON, while cluster roots were formed in the order no-N>ON>inorganic N. Intact dipeptide was transported into roots and metabolized, suggesting a role for the peptide transporter (PTR) for uptake and transport of peptides. HaPTR4, a member of subgroup II of the NRT1/PTR transporter family, which contains most characterized di- and tripeptide transporters in plants, facilitated transport of di- and tripeptides when expressed in yeast. No transport activity was demonstrated for HaPTR5 and HaPTR12, most similar to less well characterized transporters in subgroup III. The results provide further evidence that subgroup II of the NRT1/PTR family contains functional di- and tripeptide transporters. Green fluorescent protein fusion proteins of HaPTR4 and HaPTR12 localized to tonoplast, and plasma- and endomembranes, respectively, while HaPTR5 localized to vesicles of unknown identity. Grown in heathland or hydroponic culture with limiting N supply or starved of nutrients, HaPTR genes had the highest expression in cluster roots and non-cluster roots, and leaf expression increased upon re-supply of ON. It is concluded that formation of cluster roots and expression of PTR are regulated in response to N supply.

Legume crop rotation suppressed nitrifying microbial community in a sugarcane cropping soil.

Nitrifying microorganisms play an important role in nitrogen (N) cycling in agricultural soils as nitrification leads to accumulation of nitrate (NO3-) that is readily lost through leaching and denitrification, particularly in high rainfall regions. Legume crop rotation in sugarcane farming systems can suppress soil pathogens and improve soil health, but its effects on soil nitrifying microorganisms are not well understood. Using shotgun metagenomic sequencing, we investigated the impact of two legume break crops, peanut (Arachis hypogaea) and soybean (Glycine max), on the nitrifying communities in a sugarcane cropping soil. Cropping with either legume substantially increased abundances of soil bacteria and archaea and altered the microbial community composition, but did not significantly alter species richness and evenness relative to a bare fallow treatment. The ammonia oxidisers were mostly archaeal rather than bacterial, and were 24-44% less abundant in the legume cropping soils compared to the bare fallow. Furthermore, abundances of the archaeal amoA gene encoding ammonia monooxygenase in the soybean and peanut cropping soils were only 30-35% of that in the bare fallow. These results warrant further investigation into the mechanisms driving responses of ammonia oxidising communities and their nitrification capacity in soil during legume cropping.

Evolutionary conservation of a core root microbiome across plant phyla along a tropical soil chronosequence.

Culture-independent molecular surveys of plant root microbiomes indicate that soil type generally has a stronger influence on microbial communities than host phylogeny. However, these studies have mostly focussed on model plants and crops. Here, we examine the root microbiomes of multiple plant phyla including lycopods, ferns, gymnosperms, and angiosperms across a soil chronosequence using 16S rRNA gene amplicon profiling. We confirm that soil type is the primary determinant of root-associated bacterial community composition, but also observe a significant correlation with plant phylogeny. A total of 47 bacterial genera are associated with roots relative to bulk soil microbial communities, including well-recognized plant-associated genera such as Bradyrhizobium, Rhizobium, and Burkholderia, and major uncharacterized lineages such as WPS-2, Ellin329, and FW68. We suggest that these taxa collectively constitute an evolutionarily conserved core root microbiome at this site. This lends support to the inference that a core root microbiome has evolved with terrestrial plants over their 400 million year history.Yeoh et al. study root microbiomes of different plant phyla across a tropical soil chronosequence. They confirm that soil type is the primary determinant of root-associated bacterial communities, but also observe a clear correlation with plant phylogeny and define a core root microbiome at this site.

Crosstalk between sugarcane and a plant-growth promoting Burkholderia species.

Bacterial species in the plant-beneficial-environmental clade of Burkholderia represent a substantial component of rhizosphere microbes in many plant species. To better understand the molecular mechanisms of the interaction, we combined functional studies with high-resolution dual transcriptome analysis of sugarcane and root-associated diazotrophic Burkholderia strain Q208. We show that Burkholderia Q208 forms a biofilm at the root surface and suppresses the virulence factors that typically trigger immune response in plants. Up-regulation of bd-type cytochromes in Burkholderia Q208 suggests an increased energy production and creates the microaerobic conditions suitable for BNF. In this environment, a series of metabolic pathways are activated in Burkholderia Q208 implicated in oxalotrophy, microaerobic respiration, and formation of PHB granules, enabling energy production under microaerobic conditions. In the plant, genes involved in hypoxia survival are up-regulated and through increased ethylene production, larger aerenchyma is produced in roots which in turn facilitates diffusion of oxygen within the cortex. The detected changes in gene expression, physiology and morphology in the partnership are evidence of a sophisticated interplay between sugarcane and a plant-growth promoting Burkholderia species that advance our understanding of the mutually beneficial processes occurring in the rhizosphere.

Introducing BASE: the Biomes of Australian Soil Environments soil microbial diversity database.

BACKGROUND: Microbial inhabitants of soils are important to ecosystem and planetary functions, yet there are large gaps in our knowledge of their diversity and ecology. The 'Biomes of Australian Soil Environments' (BASE) project has generated a database of microbial diversity with associated metadata across extensive environmental gradients at continental scale. As the characterisation of microbes rapidly expands, the BASE database provides an evolving platform for interrogating and integrating microbial diversity and function. FINDINGS: BASE currently provides amplicon sequences and associated contextual data for over 900 sites encompassing all Australian states and territories, a wide variety of bioregions, vegetation and land-use types. Amplicons target bacteria, archaea and general and fungal-specific eukaryotes. The growing database will soon include metagenomics data. Data are provided in both raw sequence (FASTQ) and analysed OTU table formats and are accessed via the project's data portal, which provides a user-friendly search tool to quickly identify samples of interest. Processed data can be visually interrogated and intersected with other Australian diversity and environmental data using tools developed by the 'Atlas of Living Australia'. CONCLUSIONS: Developed within an open data framework, the BASE project is the first Australian soil microbial diversity database. The database will grow and link to other global efforts to explore microbial, plant, animal, and marine biodiversity. Its design and open access nature ensures that BASE will evolve as a valuable tool for documenting an often overlooked component of biodiversity and the many microbe-driven processes that are essential to sustain soil function and ecosystem services.

Nitrogen fertilizer dose alters fungal communities in sugarcane soil and rhizosphere.

Fungi play important roles as decomposers, plant symbionts and pathogens in soils. The structure of fungal communities in the rhizosphere is the result of complex interactions among selection factors that may favour beneficial or detrimental relationships. Using culture-independent fungal community profiling, we have investigated the effects of nitrogen fertilizer dosage on fungal communities in soil and rhizosphere of field-grown sugarcane. The results show that the concentration of nitrogen fertilizer strongly modifies the composition but not the taxon richness of fungal communities in soil and rhizosphere. Increased nitrogen fertilizer dosage has a potential negative impact on carbon cycling in soil and promotes fungal genera with known pathogenic traits, uncovering a negative effect of intensive fertilization.

The effect of protein supplied in the growth medium on plant pathogen resistance.

Externally supplied protein (bovine serum albumin, BSA) affects root development of Arabidopsis, increasing root biomass, root hair length, and root thickness. While these changes in root morphology may enhance access to soil microenvironments rich in organic matter, we show here that the presence of protein in the growth medium increases the plant's resilience to the root pathogen Cylindrocladium sp.

Effects of externally supplied protein on root morphology and biomass allocation in Arabidopsis.

Growth, morphogenesis and function of roots are influenced by the concentration and form of nutrients present in soils, including low molecular mass inorganic N (IN, ammonium, nitrate) and organic N (ON, e.g. amino acids). Proteins, ON of high molecular mass, are prevalent in soils but their possible effects on roots have received little attention. Here, we investigated how externally supplied protein of a size typical of soluble soil proteins influences root development of axenically grown Arabidopsis. Addition of low to intermediate concentrations of protein (bovine serum albumen, BSA) to IN-replete growth medium increased root dry weight, root length and thickness, and root hair length. Supply of higher BSA concentrations inhibited root development. These effects were independent of total N concentrations in the growth medium. The possible involvement of phytohormones was investigated using Arabidopsis with defective auxin (tir1-1 and axr2-1) and ethylene (ein2-1) responses. That no phenotype was observed suggests a signalling pathway is operating independent of auxin and ethylene responses. This study expands the knowledge on N form-explicit responses to demonstrate that ON of high molecular mass elicits specific responses.

A new species of Burkholderia isolated from sugarcane roots promotes plant growth.

Sugarcane is a globally important food, biofuel and biomaterials crop. High nitrogen (N) fertilizer rates aimed at increasing yield often result in environmental damage because of excess and inefficient application. Inoculation with diazotrophic bacteria is an attractive option for reducing N fertilizer needs. However, the efficacy of bacterial inoculants is variable, and their effective formulation remains a knowledge frontier. Here, we take a new approach to investigating diazotrophic bacteria associated with roots using culture-independent microbial community profiling of a commercial sugarcane variety (Q208(A) ) in a field setting. We first identified bacteria that were markedly enriched in the rhizosphere to guide isolation and then tested putative diazotrophs for the ability to colonize axenic sugarcane plantlets (Q208(A) ) and promote growth in suboptimal N supply. One isolate readily colonized roots, fixed N2 and stimulated growth of plantlets, and was classified as a new species, Burkholderia australis sp. nov. Draft genome sequencing of the isolate confirmed the presence of nitrogen fixation. We propose that culture-independent identification and isolation of bacteria that are enriched in rhizosphere and roots, followed by systematic testing and confirming their growth-promoting capacity, is a necessary step towards designing effective microbial inoculants.

The mixotrophic nature of photosynthetic plants.

Plants typically have photosynthetically competent green shoots. To complement resources derived from the atmospheric environment, plants also acquire essential elements from soil. Inorganic ions and molecules are generally considered to be the sources of soil-derived nutrients, and plants tested in this respect can grow with only inorganic nutrients and so can live as autotrophs. However, mycorrhizal symbionts are known to access nutrients from organic matter. Furthermore, specialist lineages of terrestrial photosynthetically competent plants are mixotrophic, including species that obtain organic nutrition from animal prey (carnivores), fungal partners (mycoheterotrophs) or plant hosts (hemi-parasites). Although mixotrophy is deemed the exception in terrestrial plants, it is a common mode of nutrition in aquatic algae. There is mounting evidence that non-specialist plants acquire organic compounds as sources of nutrients, taking up and metabolising a range of organic monomers, oligomers, polymers and even microbes as sources of nitrogen and phosphorus. Plasma-membrane located transporter proteins facilitate the uptake of low-molecular mass organic compounds, endo- and phagocytosis may enable the acquisition of larger compounds, although this has not been confirmed. Identifying the mechanisms involved in the acquisition of organic nutrients will provide understanding of the ecological significance of mixotrophy. Here, we discuss mixotrophy in the context of nitrogen and phosphorus nutrition drawing parallels between algae and plants.

Arabidopsis and Lobelia anceps access small peptides as a nitrogen source for growth.

While importance of amino acids as a nitrogen source for plants is increasingly recognised, other organic N sources including small peptides have received less attention. We assessed the capacity of functionally different species, annual and nonmycorrhizal Arabidopsis thaliana (L.) Heynh. (Brassicaceae) and perennial Lobelia anceps L.f. (Campanulaceae), to acquire, metabolise and use small peptides as a N source independent of symbionts. Plants were grown axenically on media supplemented with small peptides (2-4 amino acids), amino acids or inorganic N. In A. thaliana, peptides of up to four amino acid residues sustained growth and supported up to 74% of the maximum biomass accumulation achieved with inorganic N. Peptides also supported growth of L. anceps, but to a lesser extent. Using metabolite analysis, a proportion of the peptides supplied in the medium were detected intact in root and shoot tissue together with their metabolic products. Nitrogen source preferences, growth responses and shoot-root biomass allocation were species-specific and suggest caution in the use of Arabidopsis as the sole plant model. In particular, glycine peptides of increasing length induced effects ranging from complete inhibition to marked stimulation of root growth. This study contributes to emerging evidence that plants can acquire and metabolise organic N beyond amino acids.

Nitrate paradigm does not hold up for sugarcane.

Modern agriculture is based on the notion that nitrate is the main source of nitrogen (N) for crops, but nitrate is also the most mobile form of N and easily lost from soil. Efficient acquisition of nitrate by crops is therefore a prerequisite for avoiding off-site N pollution. Sugarcane is considered the most suitable tropical crop for biofuel production, but surprisingly high N fertilizer applications in main producer countries raise doubt about the sustainability of production and are at odds with a carbon-based crop. Examining reasons for the inefficient use of N fertilizer, we hypothesized that sugarcane resembles other giant tropical grasses which inhibit the production of nitrate in soil and differ from related grain crops with a confirmed ability to use nitrate. The results of our study support the hypothesis that N-replete sugarcane and ancestral species in the Andropogoneae supertribe strongly prefer ammonium over nitrate. Sugarcane differs from grain crops, sorghum and maize, which acquired both N sources equally well, while giant grass, Erianthus, displayed an intermediate ability to use nitrate. We conclude that discrimination against nitrate and a low capacity to store nitrate in shoots prevents commercial sugarcane varieties from taking advantage of the high nitrate concentrations in fertilized soils in the first three months of the growing season, leaving nitrate vulnerable to loss. Our study addresses a major caveat of sugarcane production and affords a strong basis for improvement through breeding cultivars with enhanced capacity to use nitrate as well as through agronomic measures that reduce nitrification in soil.

Uptake of non-pathogenic E. coli by Arabidopsis induces down-regulation of heat shock proteins.

We recently demonstrated that non-pathogenic and non-symbiotic microbes E. coli and yeast are taken up by roots and used as a source of nutrients by the plant. Although this process appears to be beneficial for the plant, the nutritional gain of microbe incorporation has to exceed the energy expense of microbe uptake and digestion, and the question remains whether the presence of microbes triggers pathogen- and other stress-induced responses. Here, we present evidence that digesting microbes is accompanied by strong down-regulation of genes linked to stress response in Arabidopsis. Genome-wide transcription analysis shows that uptake of E. coli by Arabidopsis roots is accompanied by a pronounced down-regulation of heat shock proteins. Plants up-regulate heat shock proteins in response to environmental stresses including temperature, salt, light and disease agents including microbial pathogens. The pronounced down-regulation of heat shock proteins in the presence of E. coli indicates that uptake and subsequent digestion of microbes does not induce stress. Additionally it suggests that resources devoted to stress resistance in control plants may be re-allocated to the process of microbe uptake and digestion. This observation adds evidences to the notion that uptake of microbes is an active, purposeful and intentional behavior of the plant.

DNA uptake by Arabidopsis induces changes in the expression of CLE peptides which control root morphology.

The presence of externally supplied DNA in the growth medium enhances growth of lateral roots and root hairs in Arabidopsis. This phenomenon cannot be attributed to phosphorus (P) limitation because it is independent of the plants' P status. Rather, we hypothesized that DNA triggers a currently unknown signaling pathway. Analyzing the transcriptional changes of genes induced by externally supplied DNA, we show that 7 of the 17 studied CLAVATA3/ESR-related (CLEs) genes were differentially regulated. CLEs are known peptide hormones that affect plant development including root morphology. While previous research had shown that over-expression of these CLE genes alters root morphology, changes in gene expression had not been linked to environmental triggers. The differential expression of these CLEs genes and accompanied changes of the root phenotype are indicative of a DNA-elicited signal pathway which affects root development. We conclude that DNA acts as a signaling compound which induces root proliferation in a way that would enhance the plant's ability to acquire nutrients from soil organic matter. Our study further confirms the importance of CLEs for controlling root morphology in response to specific environmental conditions, and draws attention to a novel role of DNA as a signaling compound.